The invention relates to techniques for rapidly, accurately producing an in-focus image of an object, or a cross-section thereof, wherein the effect of light signals from out-of-focus foreground and/or background light sources are mostly eliminated with regard to both statistical and systematic errors. Confocal and confocal interference microscopy are finding many applications in, for example, the life sciences, the study of biological samples, industrial inspection, and semiconductor metrology. This is because of the unique three-dimensional imaging capability of these instruments.
Perhaps the most difficult multi-dimensional imaging is encountered when the background from out-of-focus images is significantly larger than the signal from the in-focus images. Such circumstances arise frequently in the study of thick samples, particularly when working in the reflection mode in contrast to the transmission mode of confocal systems.
There are two general approaches for determining the volume properties of three-dimensional microscopic specimens. Such approaches are based on conventional microscopy and confocal microscopy. Generally, the conventional microscopy approach requires less time to acquire the data but more time to process the data for a three-dimensional image, compared to the confocal microscopy approach.
In a conventional imaging system, when a part of the object to be imaged is axially displaced from its best focus location, the image contrast decreases but the brightness remains constant so that displaced, unfocused parts of the image interfere with the view of focused parts of object.
If the system's point-spread function is known and images are obtained for each independent section of the object, known computer algorithms can be applied to such images to effectively remove the signal contributed by the out-of-focus light and produce images that contain only in-focus data. Such algorithms are of several distinct types, are referred to as "computer deconvolutions," and generally require expensive computer equipment and considerable computing time and considerable amounts of data to obtain the desired statistical accuracy.
The wide field method (WFM) (D. A. Agard and J. W. Sedat, "Three-Dimensional Analysis of Biological Specimens Utilizing Image Processing Techniques," Proc. Soc. PhotoOpt. Instrum. Eng., SPIE, 264, 110-117, 1980; D. A. Agard, R. A. Steinberg, and R. M. Stroud, "Quantitative Analysis of Electrophoretograms: A Mathematical Approach to Super-Resolution," Anal. Biochem. 111, 257-268, 1981; D. A. Agard, Y. Hiraoka, P. Shaw, and J. W. Sedat, "Fluorescence Microscopy in Three Dimensions," Methods Cell Biol. 30, 353-377, 1989; D. A. Agard, "Optical Sectioning Microscopy: Cellular Architecture in Three Dimensions," Annu. Rev. Biophys. Bioeng. 13, 191-219, 1984; Y. Hiraoka, J. W. Sedat, and D. A. Agard, "The Use of a Charge-Coupled Device for Quantitative Optical Microscopy of Biological Structures," Sci. 238, 36-41, 1987; W. Denk, J. H. Strickler, and W. W. Webb, "Two-Photon Laser Scanning Fluorescence Microscopy," Sci. 248, 73-76, 1990) uses a conventional microscope to sequentially acquire a set of images of adjacent focus planes throughout the volume of interest. Each image is recorded using a cooled charge-coupled device (CCD) image sensor (J. Kristian and M. Blouke, "Charge-coupled Devices in Astronomy," Sci. Am. 247, 67-74, 1982) and contains data from both in-focus and out-of-focus image planes.
The technique of laser computed tomography is implemented using a conventional microscope. The system discussed by S. Kawata, O. Nakamura, T. Noda, H. Ooki, K Ogino, Y. Kuroiwa, and S. Minami, "Laser Computed-Tomography Microscope," Appl. Opt. 29, 3805-3809 (1990) is based on a principal that is closely related to the technique of X-ray computed tomography, but uses three-dimensional volume reconstruction rather than two-dimensional slice reconstruction. Projected images of a thick three-dimensional sample are collected with a conventional transmission microscope modified with oblique illumination optics, and the three-dimensional structure of the interior of the sample is reconstructed by a computer. Here, the data is acquired in a time short compared to that required to process data for a three-dimensional image. In one experiment by Kawata et al., ibid., the 80.times.80.times.36-voxel reconstruction required several minutes to collect all projections and send them to a minicomputer. Approximately thirty minutes then were required for digital reconstruction of the image, in spite of utilizing a vector processor at a speed of 20 million floating point operations per second (MFLOPS).
In a conventional point or pinhole-confocal microscope, light from a point source is focused within a very small space, known as a spot. The microscope focuses light reflected from, scattered by, or transmitted through the spot onto a point detector. In a reflecting point-confocal microscope the incident light is reflected or back-scattered by that portion of the sample in the spot. Any light which is reflected or back-scattered by the sample outside of the spot is not well focused onto the detector, thus it is spread out so the point detector receives only a small portion of such reflected or back-scattered light. In a transmitting point-confocal microscope, incident light is transmitted unless it is scattered or absorbed by that portion of the sample in the spot. Generally, the point source and point detector are approximated by placing masks containing a pinhole in front of a conventional light source and a conventional detector, respectively.
Similarly, in a conventional slit-confocal microscope system, light from a line source is focused into a very narrow elongated space, which is also known as a spot. The slit-confocal microscope focuses light reflected from, scattered by or transmitted through the spot onto a line detector. The line source and line detector can be approximated using a mask with a slit in front of a conventional light source and row of conventional detectors, respectively. Alternately, a line source can be approximated by sweeping a focused laser beam across the object to be imaged or inspected.
Since only a small portion of the object is imaged by the confocal microscope, either the object to be imaged must be moved, or the source and detector must be moved, in order to obtain sufficient image data to produce a complete two-dimensional or three-dimensional view of the object. Previous slit-confocal systems have moved the object linearly in a direction perpendicular to the slit to obtain successive lines of two-dimensional image data. On the other hand, point-confocal systems having only one pinhole have to be moved in a two-dimensional manner in order to acquire two-dimensional image data and in a three-dimensional manner in order to acquire a three-dimensional set of image data. The raw image data are typically stored and later processed to form a two-dimensional cross-section or a three-dimensional image of the object that was inspected or imaged. The reduced sensitivity to out-of-focus images relative to conventional microscopy leads to improved statistical accuracy for a given amount of data and the processing operation is considerably simpler in comparison to that required when processing data obtained in conventional microscopy approach.
In a system known as the Tandem Scanning Optical Microscope (TSOM), a spiral pattern of illumination and detector pinholes are etched into a Nipkow disk so, as the disk rotates, the entire stationary object is scanned in two dimensions [cf. M. Petran and M. Hadravsky, "Tandem-Scanning Reflected-Light Microscope," J. Opt. Soc. A. 58(5), 661-664 (1968); G. Q. Xiao, T. R. Corle, and G. S. Kino, "Real-Time Confocal Scanning Optical Microscope," Appl. Phys. Lett. 53, 716-718 (1988)]. In terms of the optical processing, the TSOM is basically a single point confocal microscope with a means for efficiently scanning a two-dimensional section one point at a time.
Examples of two techniques implemented to reduce the amount of scanning required to obtain a two-dimensional image with a confocal arrangement are found in the work of H. J. Tiziani and H.-M. Uhde, "Three-Dimensional Analysis by a Microlens-Array Confocal Arrangement," Appl. Opt. 33(4), 567-572 (1994) and in the patent of P. J. Kerstens, J. R. Mandeville, and F. Y. Wu, "Tandem Linear Scanning Confocal Imaging System with Focal Volumes at Different Heights", (U.S. Pat. No. 5,248,876 issued September 1993). The microlens-array confocal arrangement of Tiziani and Uhde ibid. has out-of-focus image discrimination that is the same as using a multi-pinhole source and multi-element detector in a confocal configuration. Such a system allows for a number of points to be examined simultaneously but at a compromise in discrimination against out-of-focus images. The higher the density of microlenses, the poorer the ability of the system to discriminate against out-of-focus images, and consequently, an increase in complexity and cost of the computer deconvolutions required to produce a three-dimensional image. Further, the Tiziani and Uhde ibid. system has serious limitations in axial range. This range cannot exceed the focal length of the microlens, which is proportional to the diameter of the microlens for a given numerical aperture. Therefore, as the density of the microlenses is increased, there is an associated decrease in the permitted axial range.
The Kerstens et al., ibid. system incorporates a number of pinholes and matching pinpoint detectors in a confocal arrangement to allow for a number of points to be examined simultaneously. However, as noted in the preceding paragraph, this gain is at a compromise in discrimination against out-of-focus images and as a result an increase in complexity and cost of required subsequent computer deconvolutions. The higher the density of pinholes, the poorer the ability of the system to discriminate against out-of-focus images. The highest discrimination would be achieved when using only one pinhole.
Application of confocal microscopes to inspection of electronics was suggested in T. Zapf and R. W. Wijnaendts-van-Resandt, "Confocal Laser Microscope For Submicron Structure Measurement," Microelectronic Engineering 5, 573-580 (1986) and J. T. Lindow, S. D. Bennett, and I. R. Smith, "Scanned Laser Imaging for Integrated Circuit Metrology," SPIE, 565, 81-87 (1985). The axial discrimination provided by confocal systems make them useful in the semi-conductor manufacturing environment. For example, such systems could provide for improved inspection of height dependent features such as delamination, blisters, and thickness of structures and coatings. However, there are some problems associated with using confocal imaging systems for inspection of electronics. For example, single pinhole systems require too much time for scanning the object in two directions. Optical systems for scanning a laser beam over the object are too complex; and the spinning disk approach used in the previous TSOM resulted in alignment and maintenance problems.
The number of different depth slices required (and therefore the amount of image data collected) depends upon the range of height that must be measured, and also upon the desired height resolution and performance of the optical system. For typical electronics inspection, images of 10 to 100 different depth slices would be required. Furthermore, data in several color bands may be required to differentiate materials. In confocal imaging systems, a separate two-dimensional scan is required for each desired elevation. If data for multiple color bands is desired, then multiple two-dimensional scans at each elevation are required. By shifting the focus level, similar data can be obtained from adjacent planes and a three-dimensional intensity data set can be acquired.
Thus, none of the prior art confocal microscopy systems can be configured for rapid and/or reliable three-dimensional tomographic imaging, especially in the field of inspection or imaging.
Although the confocal approach is more straightforward and works better, for example in confocal fluorescence work, when the concentration of stained structure is high, the conventional microscopy approach still has several practical advantages. The most important of these is that the latter can utilize dyes that are excited in the ultraviolet (UV) range and these often seem more robust and efficient than those excited in the visible range. Although, a UV laser can be incorporated as the light source of a confocal microscope [M. Montag, J. Kululies, R. Jorgens, H. Gundlach, M. F. Trendelenburg, and H. Spring, "Working with the Confocal Scanning UV-Laser Microscope: Specific DNA Localization at High Sensitivity and Multiple-Parameter Fluorescence," J. Microsc(Oxford) 163 (Pt. 2), 201-210, 1991; K. Kuba, S.-Y. Hua, and M. Nohmi, "Spatial and Dynamic Changes in Intracellular Ca.sup.2+ Measured by Confocal Laser-Scanning Microscopy in Bullfrog Sympatetic Ganglion Cells," Neurosci. Res. 10, 245-259, 1991; C. Bliton, J. Lechleiter and D. E. Clapham, "Optical Modifications Enabling Simultaneous Confocal Imaging With Dyes Excited by Ultraviolet- and Visible-Wavelength Light," J. Microsc. 169(Pt. 1), 15-26, 1993], or UV dyes can be excited with infrared (IR) light using the "two photon" technique (W. Denk, et al., ibid.), these techniques involve considerable expense and practical difficulty.
Furthermore, the cooled CCD detectors used in conventional microscopy systems collect the data in parallel rather than serially, as does the photomultiplier (PMT) in a confocal microscopy system. As a result, if the CCD can be made to read out more rapidly without degrading its performance, the three-dimensional data recording rate of the conventional microscopy system may prove to be significantly higher than that of the confocal microscopy system, even though the time needed for computer deconvolution computations means that there might be an additional delay before the data could be actually viewed as three-dimensional image.
The signal-to-noise ratio in relation to statistical accuracy must also be considered when making a choice between a CCD detector used to record in parallel a two-dimensional data array and a slit or pinhole confocal microscope. The well capacity of a two-dimensional CCD pixel is of the order of 200,000 electrons. This limits the statistical accuracy that can be achieved in a single exposure as compared to that achievable with other photoemissive detectors such as PMT's or photovoltaic devices. Consequently, for those applications where the out-of-focus background contributions are significantly larger than the in-focus image signals, consideration of the signal-to-noise ratio may lead to the conclusion that a one-dimensional parallel recording of data in a slit confocal microscope will perform better than a two-dimensional recording of data in a standard microscope configuration or a point by point recording of data in a single pinhole confocal microscope will perform better than a one-dimensional parallel recording of data in a slit confocal microscope, all other considerations being equal.
When the consideration of statistical accuracy as measured by the signal-to-noise ratio influences the selection of a system such as a slit confocal microscope over a standard microscope, or a single pinhole confocal microscope over a slit confocal microscope, the residual signals from the out-of-focus images for the system chosen can be comparable to or larger than the in-focus signals. Such is the case for example when examining deep into biological samples at optical wavelengths where scattering of optical radiation dominates over absorption. In this case, one is left with the need for a lengthy computer deconvolution, i.e. long compared to the time required to acquire the data. Note that this is in general true for the single pinhole confocal microscope as well as the slit confocal microscope when looking for an in-focus image signal that is much smaller than the residual out-of-focus image signals.
Although it is easier to accurately digitize the signal from a CCD detector than from a PMT (J. B. Pawley, "Fundamental and Practical Limits in Confocal Light Microscopy," Scanning 13, 184-198, 1991), the PMT is a single device that can be accurately characterized, whereas the CCD is actually a large array of discrete detectors and additional noise is associated with correcting for the pixel-to-pixel variations in sensitivity and offset that characterize its operation (Y. Hiraoka, et al., ibid.; J. E. Wampler and K. Kutz, "Quantitative Fluorescence Microscopy Using Photomultiplier Tubes and Imaging Detectors," Methods Cell Biol. 29, 239-267, 1989; Z. Jericevic, B. Wiese, J. Bryan, and L. C. Smith, "Validation of an Imaging System: Steps to Evaluate and Validate a Microscope Imaging System for Quantitative Studies," Methods Cell Biol. 30, 47-83, 1989).
It should be noted that the above distinction between the photodetectors used in the two methods of three-dimensional microscopy should not be considered to be complete, because the cooled CCD detector is the most suitable photodetector for those confocal microscopes that accomplish the scanning function by using holes in a spinning disk (Petran, et al., ibid.; Xiao, et al., ibid.).
Another technique known as "optical coherence-domain reflectometry" (OCDR) has been used to obtain information about the three-dimensional properties of a system. This method is described in the following articles: (1) "Optical Coherence-Domain Reflectometry: A New Optical Evaluation Technique," by R. C. Youngquist, S. Carr, and D. E. N. Davies, Opt. Lett. 12(3), 158-160 (1987); (2) "New Measurement System for Fault Location in Optical Waveguide Devices Based on an Interferometric Technique," K. Takada, I. Yokohama, K. Chida, and J. Noda, Appl. Opt. 26(9), pp. 1603-1606 (1987); (3) "Guided-Wave Reflectometry with Micrometer Resolution," B. L. Danielson and C. D. Whittenberg, Appl. Opt. 26(14), 2836-2842 (1987). The OCDR method differs from the coherent optical time domain reflectometry (OTDR) technique in that instead of a pulsed light source one uses a broadband continuous-wave source with a short coherence length. The source beam enters an interferometer in which one arm has a movable mirror, with the reflected light from this mirror providing a reference beam, and the other arm contains the optical system being tested. The interference signal in the coherently mixed reflected light from the two arms is detected by the usual heterodyne method and yields the desired information about the optical system.
The heterodyne detection of the backscattered signals in the OCDR technique is accomplished by the method of "white-light interferometry," in which the beam is split into the two arms of an interferometer, reflected by the adjustable mirror and the backscattering site, and coherently recombined. This method utilizes the fact that interference fringes will appear in the recombined beam only when the difference in the optical path length between the two arms is less than the coherence length of the beam. The OCDR systems described in references (1) and (3) above make use of this principle, and reference (3) shows interferograms of fiber gaps in test systems obtained by scanning the adjustable mirror and measuring the strength of the recombined signal. Reference (1) also describes a modified method in which the mirror in the reference arm oscillates at a controlled frequency and amplitude, causing a Doppler shift in the reference signal, and the recombined signal is fed into a filtering circuit to detect the beat frequency signal.
Another variation of this technique is illustrated in reference (2), in which the reference arm mirror is at a fixed position and the difference in optical path lengths in the two arms may exceed the coherence length. The combined signal is then introduced into a second Michelson interferometer with two mirrors, one fixed in position and the other being moveable. This moveable mirror is scanned and the difference in path length between the arms of the second interferometer compensates for the delay between the backscattered and reference signals at discrete positions of the moveable mirror corresponding to the scattering sites. In practice, an oscillating phase variation at a definite frequency is imposed on the signal from the backscattering site by means of a piezoelectric transducer modulator in the fiber leading to this site. The output signal from the second Michelson interferometer is fed to a lock-in amplifier, which detects the beat frequency signal arising from both the piezoelectric transducer modulation and the Doppler shift caused by the motion of the scanning mirror. This technique has been used to measure irregularities in glass waveguides with a resolution as short as 15 .mu.m ["Characterization of Silica-Based Waveguides with a Interferometric Optical Time-Domain Reflectometry System Using a 1.3-.mu.m-Wavelength Superluminescent Diode," K. Takada, N. Takato, J. Noda, and Y. Noguchi, Opt. Lett. 14(13), 706-708 (1989)].
Another variation of the OCDR is the dual-beam partial coherence interferometer (PCI) which has been used to measure the thickness of fundus layers in the eye ["Measurement of the Thickness of Fundus Layers by Partial Coherence Tomography," by W. Drexler, C. K. Hitzenberger, H. Sattmann, and A. F. Fercher, Opt. Eng. 34(3), 701-710 (1995)]. In the PCI used by Drexler, et al., an external Michelson interferometer splits a light beam of high spatial coherence but very short coherence length of 15 .mu.m into two parts: the reference beam (1) and the measurement beam (2). At the interferometer exit, these two components are combined again to form a coaxial dual beam. The two beam components, which have a path difference of twice the interferometer arm length difference, illuminate the eye and are reflected at several intraocular interfaces, which separate media of different refractive index. Therefore each beam component (1 and 2) is further split into subcomponents by reflection at these interfaces. The reflected subcomponents are superimposed on a photodetector. If the optical distance between two boundaries within the eye equals twice the interferometer arm length difference, there are two subcomponents that will travel over the same total path length and will consequently interfere. Each value of the interferometer arm length difference where an interference pattern is observed, is equal to an intraocular optical distance. Provided that there is no other strong reflection nearby, the absolute position of these interfaces can be determined in vivo with a precision of 5 .mu.m. However, the PCI suffers from limitations due to motion of the object during the time required for the 3-D scanning.
Another variation of the OCDR called optical coherent tomography (OCT) has been reported for in vivo retinal imaging by E. A. Swanson, J. A. Izatt, M. R. Hee, D. Huang, C. P. Lin, J. S. Schuman, C. A. Puliafito, and J. G. Fujimoto, "In Vivo Retinal Imaging by Optical Coherence Tomography," Opt. Lett. 18(21), 1864-1866 (1993), and E. A. Swanson, D. Huang, J. G. Fujimoto, C. A Puliafito, C. P. Lin, and J. S. Schuman, "Method and Apparatus for Optical Imaging with Means for Controlling the Longitudinal Range of the Sample," U.S. Pat. No. 5,321,501, issued Jun. 14, 1994. The above referenced patent describes a method and apparatus for performing optical imaging on a sample wherein longitudinal scanning or positioning in the sample is provided by either varying relative optical path lengths for an optical path leading to the sample and to a reference reflector, or by varying an optical characteristic of the output from an optical source applied to the apparatus. Transverse scanning in one or two-dimensions is provided on the sample by providing controlled relative movement between the sample and a probe module in such direction and/or by steering optical radiation in the probe module to a selected transverse position. The reported spatial resolution is &lt;20 .mu.m with a high sensitive (100 dB dynamic range). However the OTC suffers from limitations due to motion of the object during the time required for the three-dimensional scanning.
Optical interferometric profilers are widely used for three-dimensional profiling of surfaces when noncontact methods are required. These profilers typically use phase-shifting interferometric (PSI) techniques and are fast, accurate, and repeatable, but suffer from the requirement that the surface be smooth relative to the mean wavelength of the light source. Surface discontinuities greater than a quarter-wavelength (typically 150 nm) cannot be unambiguously resolved with a single-wavelength measurement because of the cyclic nature of the interference. Multiwavelength measurements can extend this range, but the constraints imposed on wavelength accuracy and environmental stability can be severe (U.S. Pat. No. 4,340,306 issued Jul. 20, 1982 to N. Balasubramanian entitled "Optical System for Surface Topography Measurement.")
Profilers based on scanning white-light interferometry (SWLI) overcome many of the limitations of conventional PSI profilers for the measurement of rough or discontinuous surfaces. A number of articles describe this technique in detail [cf. Refs. 2-7 in L. Deck and P. de Groot, Appl. Opt. 33(31), 7334-7338 (1994)]. Typically these profilers record the position of a contrast reference feature (i.e., peak contrast or peak fit) for each point in the field of view while axially translating one arm of an equal-path interferometer illuminated with a broadband source. A common problem with this technique is the enormous amount of computation required for calculating the contrast for each point in real time. Often the contrast calculation alone is insufficiently precise because of the discrete sampling interval, forcing either an increase in the sampling density or incorporating an interpolation technique, both of which further slow the acquisition process. The Coherence Probe Microscope (CPM) is an example of this class of profiler [U.S. Pat. No. 4,818,110 issued Apr. 4, 1989 to M. Davidson entitled "Method and Apparatus of Using a Two Beam Interference Microscope for Inspection of Integrated Circuits and the Like"; M. Davidson, K. Kaufman, I. Mazor, and F. Cohen, "An Application of Interference Microscope to Integrated Circuit Inspection and Metrology," SPIE, 775, 233-247 (1987); U.S. Pat. No. 5,112,129 issued May 12, 1992 to M. Davidson, K. Kaufman, and I. Mazor entitled "Method of Image Enhancement for the Coherence Probe Microscope with Applications to Integrated Circuit Metrology."]. Profilers in general and the CPM in particular are not able to work with three-dimensional objects, have the background typical of a conventional interference microscopy, are sensitive to vibrations, and require computer intensive analysis.
Profilers based on triangulation also overcome many of the limitations of conventional PSI profilers but suffer from reduced height and lateral space resolution and have a large background form out-of-images. An application of this technique is found in the paper entitled "Parallel Three-Dimensional Sensing by Color-Coded Triangulation" by G. Hausler and D. Ritter, Appl. Opt., 32(35), 7164-7169 (1993). The method used by Hausler and Ritter, ibid., is based on the following principle: a color spectrum of a white-light source is imaged onto the object by illumination from one certain direction. The object is observed by a color TV camera from a direction of observation which is different from the direction of illumination. The color (hue) of each pixel is a measure of its distance from a reference plane. The distance can be evaluated by the three(red-green-blue) output channels of a charge coupled device (CCD) camera and this evaluation can be implemented within TV real time. However, the resolution in height and in one lateral spatial dimension is considerably reduced below that achieved with PSI and SWLI, there is a large background, and the triangulation profiler has the noise characteristics of non-interferometric measurement techniques. In addition, the triangulation profiler is limited to surface profiling.
One of the problems encountered in white-light interferometry (WLI) is the problem of phase ambiguities. A profilometry method that has been received attention with respect to the phase ambiguity problem is the dispersive interferometric profilometer (DIP) proposed by J. Schwider and L. Zhou in a paper entitled "Dispersive Interferometric Profilometer," Opt. Lett. 19(13), 995-997 (1994). A similar approach for WLI has also been reported by U. Schnell, E. Zimmermann, and R. Dandliker in an article entitled "Absolute Distance Measurement With Synchronously Sampled White-Light Channelled Spectrum Interferometry," Pure Appl. Opt. 4, 643-651 (1995).
In general, the phase ambiguity problem can be completely avoided with the use of DIP. In the DIP apparatus, a parallel beam of a white-light source perpendicularly impinges upon the real wedge of a Fizeau interferometer in front of an apochromatic microscope objective. The Fizeau interferometer is formed by the inner surface of the reference plate and the object surface. Then the light is reflected back onto the slit of a grating spectrometer, which disperses the sofar invisible fringe pattern and projects the spectrum onto a linear array detector. On the detector each point of the surface selected by the slit of the spectrometer furnishes a dispersed spectrum of the air gap in the Fizeau interferometer. The fringe patterns can be evaluated by use of Fourier-transform and filtering methods to obtain the phase information from the intensity distribution of a wedge-type interferogram.
Although the phase ambiguity problem can be avoided with the use of DIP, DIP is not suitable in applications requiring the examination of three-dimensional objects. This is a consequence of the intrinsic relatively large background produced in DIP from out-of-focus images. The background problem is comparable to the background problem faced when trying to produce three-dimensional images using standard interference microscopy.
An apparatus and method for making spectrally-resolved measurements of light reflected, emitted or scattered from a specimen was disclosed by A. E. Dixon, S. Damaskinos, and J. W. Bowron in U.S. Pat. No. 5,192,980 issued Mar. 9, 1993 and entitled "Apparatus and Method for Spatially- and Spectrally-Resolved Measurements". In one set of embodiments of the apparatus and method of Dixon et al., properties of a specimen are characterized in terms of the intensity of light reflected, emitted or scattered from the specimen wherein the apparatus and method are comprised of non interferometric, non confocal type with a dispersive element preceding the detector. This set of embodiments of Dixon et al. have a large background from out-of-focus images intrinsic to the standard microscope, the set of embodiments being of the non confocal type.
The apparatus and method of Dixon et al. also includes a non interferometric confocal embodiment which permits measurements with reduced background. However the limitation to making intensity measurements for the confocal embodiment as well as for the non confocal embodiments, a consequence of using a non interferometric technique, poses serious limitations on the information about the specimen that can be acquired from reflected or scattered light. Intensity measurements yield information about the square of the magnitude of an amplitude of light reflected or scattered by the specimen with the consequence that information about the phase of the amplitude of reflected or scattered light is lost. The apparatus and method of Dixon et al. Further includes an embodiment which incorporates a Fourier Transform spectrometer in a non confocal imaging system. The Fourier Transform spectrometer embodiment of Dixon et al. has the disadvantage of a large background from out-of-focus images intrinsic to nonconfocal imaging systems.
Apparatus for making simultaneous multiple wavelength measurements with a non-interferometric, confocal imaging system has been disclosed by G. Xiao in U.S. Pat. No. 5,537,247 issued July 1996 and entitled "Single Aperture Confocal Imaging System". The apparatus of Xiao is comprised of a confocal scanning imaging system which utilizes only one aperture for both the incident light from the light source and return light from the object and a series of beam splitters and optical wavelength filters to selectively direct return light of differing wavelengths to a series of detectors, respectively. The Xiao apparatus has an advantage of making simultaneous measurements at different wavelengths and the merits of a confocal imaging system with respect to reduced background from out-of-focus images. However the limitation to making intensity measurements, a consequence of using a non interferometric technique, poses serious limitations on the information about the specimen that can be acquired from reflected or scattered light. Intensity measurements yield information about the square of the magnitude of an amplitude of light reflected or scattered by the specimen with the consequence that information about the phase of the amplitude of reflected or scattered light is lost.
It was pointed out in a paper by G. Q. Xiao, T. R. Corle, and G. S. Kino entitled "Real-time Confocal Scanning Optical Microscope," Appl. Phys. Lett., 53(8), 716-718 (1988) that when using white light in a confocal microscope, the chromatic aberrations of the objective lens ensures that images from different heights in the specimen are all present and all in focus but at different colors. Xiao et al. demonstrated this by producing images of a silicon integrated circuit at four different wavelengths. H. J. Tiziani and H.-M. Uhde described in a paper entitled "Three-Dimensional Image Sensing by Chromatic Confocal Microscopy," Appl. Opt., 33(10), 1838-1843 (1994) a white light, non interferometric, confocal microscope in which chromatic aberration was deliberately introduced into the microscope objective for the purpose of obtaining height information without physically scanning the object. A camera with black-and-white film sequentially combines, with three selected chromatic filters, intensity and tone of color of each object point. Although confocal microscopes are used in both of the works described by Xiao et al. and Tiziani and Uhde and therefore have reduced background from out-of-focus images, they are limited to making intensity measurements. The limitation to making intensity measurements, a direct consequence of using a non interferometric technique, poses serious limitations on the information about the specimen that can be acquired from reflected or scattered light as noted in reference to the patents by Dixon et al. and Xiao.
An interference microscope has been described in papers by G. S. Kino and S. C. Chim, "Mirau Correlation Microscope," Appl. Opt., 26(26), 3775-3783 (1990) and S. S. C. Chim and G. S. Kino, "Three-Dimensional Image Realization in Interference Microscopy," Appl. Opt., 31(14), 2550-2553 (1992) which is based on a Mirau interferometer configuration. The apparatus of Kino and Chim employs an interferometric, non confocal microscope with a spatially and temporally incoherent light source and uses as the detected output the correlation signal between the beams reflected from the object and from a mirror, respectively. It is possible with the apparatus of Kino and Chim to measure both amplitude and phase of the beam reflected from the object. However, the interferometric apparatus of Kino and Chim has the disadvantage of a serious background problem, the level of background from out-of-focus images being typical of that found in a standard interference, nonconfocal microscopy system.
An interferometric apparatus has been disclosed by A. Knuttel in U.S. Pat. No. 5,565,986 issued Oct. 15, 1996 and entitled "Stationary Optical Spectroscopic Imaging in Turbid Objects by Special Light Focusing and Signal Detection of Light with Various Optical Wavelengths" to obtain a spectroscopic image of an object, displaying both spatial resolution in a lateral direction and a field of view in a depth direction. The apparatus described by Knuttel has a nonconfocal imaging system and typically includes a dispersive optical element in an arm of an interferometer and a chromatic object lens. The dispersive element makes it possible to record information about the scattered light amplitude at different optical wavelengths, the use of an interferometer makes it possible to record information about the magnitude and phase of the amplitude of reflected or scattered light, and the use of a chromatic object lens makes it possible to record information about a field of view in a depth direction. However, the interferometric apparatus of Knuttel has a serious background problem, the level of the background being typical of that found in a standard interference, nonconfocal microscopy system.
One of the primary objectives of an embodiment of the apparatus of Knuttel was to be able to image simultaneously two regions of an object separated in a depth dimension by using two different orders of a chromatic object lens comprised in part of a zone plate. As a consequence, the signals recorded by the detector of this embodiment are comprised of superimposed images from the two separated depth positions in the object. Therefore, in addition to the presence of a high background from out-of-focus images as previously noted, a complex inversion calculation must be performed by the computer to extract the image for a given depth from the superimposed in focus images. There is a serious problem encountered with the type of inversion calculation required for superimposed images as acquired with the referenced embodiment of Knuttel: the results of the inversion calculations are relatively accurate near the surface of the object but rapidly degrade as the depth in the sample increases. This problem is generally not encountered in inversion calculations where there is only one point of the object in-focus at the detector.
The above cited background problem encountered in interference microscopy is reduced in an interference version of the confocal microscope described by D. K. Hamilton and C. J. R. Sheppard in an article entitled "A Confocal Interference Microscope", Optica Acta, 29(12), 1573-1577 (1982). The system is based on the confocal microscope in which the object is scanned relative to a focused laser spot, the laser spot being arranged to coincide with the back-projected image of a point detector. An interference form of the reflection confocal microscope is based on a Michelson interferometer in which one beam is focused onto the object. This system has the important property of a reduced background from out-of-focus images intrinsic to confocal microscopy systems. However, the confocal interference microscope of Hamilton and Sheppard, ibid., measures the reflected signal at only one point at a time in a three-dimensional object. The scanning of the object one point at a time also makes the system sensitive to sample motion unrelated to the scan during the required data acquisition.
A major component that is important in the effective utilization of high-performance computers is memory. Because of the huge data storage requirements of these instruments, compact, low-cost, very high-capacity, high-speed memory devices are needed to handle the high data volume afforded by parallel computing. Such data storage requirements may be provided by a three-dimensional memory.
In a two-dimensional memory, the maximum theoretical storage density (proportional to 1/.lambda..sup.2) is of the order of 3.5.times.10.sup.8 bits/cm.sup.2 for .lambda.=532 nm, whereas in a three-dimensional memory the maximum storage density is of the order of 6.5.times.10.sup.12 bits/cm.sup.3. These maximum values represent upper limits to the storage capacity when using a single bit binary format at each memory site. These upper limits can be increased by using a recording medium where different levels of amplitude or amplitude and phase information are recorded. Holographic recording in phase-recording media is an example of the latter mode.
In the different modes of recording, the mode of single bit binary format, amplitude in base N format or amplitude and phase in (base N).times.(base M) format, at each memory site, the size of a voxel at a memory site that can be used, and therefore storage density, is limited by the signal-to-noise ratio that can be obtained, the signal-to-noise ratio generally being inversely proportional to the volume of the voxel. In particular, for the amplitude or amplitude and phase recording modes, the number of independent pieces of y information that can be stored in a voxel is also limited by the signal-to-noise ratio that can be obtained.
What is needed is a system that combines a sensitivity of image data to out-of-focus images that is reduced below that inherent in prior art confocal and confocal interference microscopy, the reduced sensitivity of the image data to out-of-focus images being with respect to both systematic and statistical errors; a reduced requirement of computer deconvolutions associated with reduced sensitivity to out-of-focus images; the potential for high signal-to-noise ratios intrinsic to confocal interference microscopy systems; capacity to record in parallel the data for an axial or transverse direction; and the potential to measure the complex amplitude of the scattered and/or the reflected light beam.